Setting the chaperonin timer: a two-stroke, two-speed, protein machine

In a study of the timing mechanism of the chaperonin nanomachine we show that the hemicycle time (HCT) is determined by the mean residence time (MRT) of GroES on the cis ring of GroEL. In turn, this is governed by allosteric interactions within the trans ring of GroEL. Ligands that enhance the R (relaxed) state (residual ADP, the product of the previous hemicycle, and K⁺) extend the MRT and the HCT, whereas ligands that enhance the T (taut) state (unfolded substrate protein, SP) decrease the MRT and the HCT. In the absence of SP, the chaperonin machine idles in the resting state, but in the presence of SP it operates close to the speed limit, set by the rate of ATP hydrolysis by the cis ring. Thus, the conformational states of the trans ring largely control the speed of the complete chaperonin cycle.     

GroEL/GroES cycling: ATP binds to an open ring before substrate protein favoring protein binding and production of the native state

The GroEL/GroES reaction cycle involves steps of ATP and polypeptide binding to an open GroEL ring before the GroES encapsulation step that triggers productive folding in a sequestered chamber. The physiological order of addition of ATP and nonnative polypeptide, typically to the open trans ring of an asymmetrical GroEL/GroES/ADP complex, has been unknown, although there have been assumptions that polypeptide binds first, allowing subsequent ATP-mediated movement of the GroEL apical domains to exert an action of forceful unfolding on the nonnative polypeptide. Here, using fluorescence measurements, we show that the physiological order of addition is the opposite, involving rapid binding of ATP, accompanied by nearly as rapid apical domain movements, followed by slower binding of nonnative polypeptide. In order-of-addition experiments, approximately twice as much Rubisco activity was recovered when nonnative substrate protein was added after ATP compared with it being added before ATP, associated with twice as much Rubisco protein recovered with the chaperonin. Furthermore, the rate of Rubisco binding to an ATP-exposed ring was twice that observed in the absence of nucleotide. Finally, when both ATP and Rubisco were added simultaneously to a GroEL ring, simulating the physiological situation, the rate of Rubisco binding corresponded to that observed when ATP had been added first. We conclude that the physiological order, ATP binding before polypeptide, enables more efficient capture of nonnative substrate proteins, and thus allows greater recovery of the native state for any given round of the chaperonin cycle.     

The endocochlear potential depends on two K+ diffusion potentials and an electrical barrier in the stria vascularis of the inner ear

An endocochlear potential (EP) of +80 mV is essential for audition. Although the regulation of K(+) concentration ([K(+)]) in various compartments of the cochlear stria vascularis seems crucial for the formation of the EP, the mechanism remains uncertain. We have used multibarreled electrodes to measure the potential, [K(+)], and input resistance in each compartment of the stria vascularis. The stria faces two fluids, perilymph and endolymph, and contains an extracelluar compartment, the intrastrial space (IS), surrounded by two epithelial layers, the marginal cell (MC) layer and that composed of intermediate and basal cells. Fluid in the IS exhibits a low [K(+)] and a positive potential, called the intrastrial potential (ISP). We found that the input resistance of the IS was high, indicating this space is electrically isolated from the neighboring extracellular fluids. This arrangement is indispensable for maintaining positive ISP. Inhibiting the K(+) transporters of the stria by anoxia, ouabain, or bumetanide caused the [K(+)] of the IS to increase and the intracellular [K(+)] of MCs to decrease, reducing both the ISP and the EP. Calculations indicate that the ISP represents the K(+) diffusion potential across the apical membranes of intermediate cells through Ba(2+)-sensitive K(+) channels. The K(+) diffusion potential across the apical membranes of MCs also contributes to the EP. Because the EP depends on two K(+) diffusion potentials and an electrical barrier in the stria vascularis, interference with any of these elements can interrupt hearing.     

ATP-sensitive potassium channel (K(ATP))-dependent regulation of cardiotropic viral infections

The effects of the cellular environment on innate immunity remain poorly characterized. Here, we show that in Drosophila ATP-sensitive potassium channels (K(ATP)) mediate resistance to a cardiotropic RNA virus, Flock House virus (FHV). FHV viral load in the heart rapidly increases in K(ATP) mutant flies, leading to increased viremia and accelerated death. The effect of K(ATP) channels is dependent on the RNA interference genes Dcr-2, AGO2, and r2d2, indicating that an activity associated with this potassium channel participates in this antiviral pathway in Drosophila. Flies treated with the K(ATP) agonist drug pinacidil are protected against FHV infection, thus demonstrating the importance of this regulation of innate immunity by the cellular environment in the heart. In mice, the Coxsackievirus B3 replicates to higher titers in the hearts of mayday mutant animals, which are deficient in the Kir6.1 subunit of K(ATP) channels, than in controls. Together, our data suggest that K(ATP) channel deregulation can have a critical impact on innate antiviral immunity in the heart.     

Remodeling of outward K+ currents in pressure-overload heart failure

Objectives: Outward K⁺ currents are critical determinants of action potential repolarization and the site of action of a number of electrophysiologically active drugs. Further, expression and processing of the channels underlying these currents is altered in heart disease. Here, we investigated the native transmural gradient of outward K⁺ currents in murine left ventricle (LV) and delineated disease‐related remodeling of these currents in heart failure (HF). Methods: Pressure‐overload heart failure was induced in mice by thoracic aortic constriction. Outward K⁺ currents were recorded using the whole‐cell patch clamp technique in acutely dissociated ventricular myocytes. Results: Unambiguous gradients of outward K⁺ current density and Kv4.2 protein abundance were observed across the wall of the LV, with significantly larger current density and protein levels in subepicardial (SEP) myocytes, compared with subendocardial (SEN) myocytes. Voltage dependences of current activation and inactivation were similar in SEP and SEN myocytes. In failing LV, however, outward K⁺ current density was significantly decreased in SEP but not in SEN cells leading to elimination of the native transmural gradient. In failing LV, the voltage dependences of K⁺ current activation and inactivation were not altered. However, current inactivation (decay) was significantly accelerated and recovery from inactivation was significantly slowed. Consistent with this, Western blot analysis revealed a decrease in KChIP2 protein abundance in failing LV. Conclusions: This is the first report of HF‐related remodeling of outward K⁺ currents in murine LV. Similar to humans, disease‐related remodeling occurs differentially across the murine ventricular wall, leading to loss of the native gradient of repolarization. Together with slowed recovery from inactivation, these alterations likely promote abnormal impulse conduction, a major proarrhythmic mechanism.     

Domain-swapped chain connectivity and gated membrane access in a Fab-mediated crystal of the human TRAAK K+ channel

TRAAK (TWIK-related arachidonic acid-stimulated K⁺ channel, K2P4.1) K⁺ ion channels are expressed predominantly in the nervous system to control cellular resting membrane potential and are regulated by mechanical and chemical properties of the lipid membrane. TRAAK channels are twofold symmetric, which precludes a direct extension of gating mechanisms that close canonical fourfold symmetric K⁺ channels. We present the crystal structure of human TRAAK in complex with antibody antigen-binding fragments (Fabs) at 2.75-Å resolution. In contrast to a previous structure, this structure reveals a domain-swapped chain connectivity enabled by the helical cap that exchanges two opposing outer helices 180° around the channel. An unrelated conformational change of an inner helix seals a side opening to the membrane bilayer and is associated with structural changes around the K⁺-selectivity filter that may have implications for mechanosensitivity and gating of TRAAK channels.     

K+-induced dilation of hamster cremasteric arterioles involves both the Na+/K+-ATPase and inward-rectifier K+ channels

OBJECTIVE: The mechanism by which elevated extracellular potassium ion concentration ([K+]o) causes dilation of skeletal muscle arterioles was evaluated. METHODS: Arterioles (n = 111) were hand-dissected from hamster cremaster muscles, cannulated with glass micropipettes and pressurized to 80 cm H2O for in vitro study. The vessels were superfused with physiological salt solution containing 5 mM KCl, which could be rapidly switched to test solutions containing elevated [K+]o and/or inhibitors. The authors measured arteriolar diameter with a computer-based diameter tracking system, vascular smooth muscle cell membrane potential with sharp micropipettes filled with 200 mM KCl, and changes in intracellular Ca2+ concentration ([Ca2+]i) with Fura 2. Membrane currents and potentials also were measured in enzymatically isolated arteriolar muscle cells using patch clamp techniques. The role played by inward rectifier K+ (KIR) channels was tested using Ba2+ as an inhibitor. Ouabain and substitution of extracellular Na+ with Li+ were used to examine the function of the Na+/K+ ATPase. RESULTS: Elevation of [K+]o from 5 mM up to 20 mM caused transient dilation of isolated arterioles (27 +/- 1 microm peak dilation when [K+]o was elevated from 5 to 20 mM, n = 105, p <.05). This dilation was preceded by transient membrane hyperpolarization (10 +/-1 mV, n = 23, p <.05) and by a fall in [Ca2+]i as indexed by a decrease in the Fura 2 fluorescence ratio of 22 +/- 5% (n = 4, p <.05). Ba(2+) (50 or 100 microM) attenuated the peak dilation (40 +/- 8% inhibition, n = 22) and hyperpolarization (31 +/- 12% inhibition, n = 7, p <.05) and decreased the duration of responses by 37 +/-11% (n = 20, p < 0.05). Both ouabain (1 mM or 100 microM) and replacement of Na+ with Li+ essentially abolished both the hyperpolarization and vasodilation. CONCLUSIONS: Elevated [K+]o causes transient vasodilation of skeletal muscle arterioles that appears to be an intrinsic property of the arterioles. The results suggest that K+-induced dilation involves activation of both the Na+/K+ ATPase and KIR channels, leading to membrane hyperpolarization, a fall in [Ca2+]i, and culminating in vasodilation. The Na+/K+ ATPase appears to play the major role and is largely responsible for the transient nature of the response to elevated [K+]o, whereas KIR channels primarily affect the duration and kinetics of the response.     

Accelerated inactivation of voltage-dependent K+ outward current in cardiomyocytes from thyroid hormone receptor alpha1-deficient mice

INTRODUCTION: Thyroid hormone affects the electrophysiologic properties of the heart. It is not known which of the different subtypes of thyroid hormone receptors mediate these effects. METHODS AND RESULTS: Using standard patch-clamp techniques, we studied time- and voltage-dependent properties of depolarization-activated K+ currents in ventricular heart cells isolated from mice lacking the thyroid hormone receptor alpha1 (TR alpha1) and compared these currents with those in respective wild-type cells. In both groups of cells, the time course of current decay could be described by two inactivating exponential components and a sustained current component. In TR alpha1-deficient cells, the total inactivation time course was accelerated due to both increase of the relative contribution of the fast component and shortening of the slow time constant. The peak amplitude of the total current was not altered. The main component of steady-state inactivation of the voltage-dependent K+ outward current was shifted to more hyperpolarized voltages by 7 mV in TR alpha1-deficient cells compared with that in wild-type cells. Under current-clamp conditions, action potential duration at 90% repolarization was prolonged in TR alpha1-deficient cells compared with that in wild-type cells by 3.6 msec. CONCLUSION: The resulting acceleration of the total inactivation time course is proposed to contribute to action potential prolongation and thus to the increased QTend-time observed previously on ECG of TR alpha1-deficient mice.     

西沙比利对HERG基因表达的快速激活延迟整流钾通道电流及蛋白的影响

目的评价西沙比利对转染HERG基因表达的快速激活延迟整流钾电流(IKr)和蛋白的影响,探讨其致心律失常的机制。方法使用脂质体介导的瞬时转染法把野生型HERG基因转染进人胚肾细胞(HEK293),采用全细胞膜片钳技术评价西沙比利对IKr通道电流和动力学的影响;使用G418筛选出稳定转染HERG的细胞,并用西沙比利进行干预,应用免疫蛋白印迹技术评价药物对蛋白的影响。结果西沙比利对IKr通道的刺激电流(IHERG)和尾电流(Itail)具有浓度和电压依赖性抑制作用,半数最大抑制浓度分别14.5和3.9nmol/L。西沙比利使IHERG和Itail的最大峰值电位前移,但不改变激活电位;使激活曲线左移并加快通道的失活,但不改变通道失活后的恢复时间;西沙比利对转染后的HEK293细胞上的IKr通道蛋白无明显抑制。结论西沙比利通过作用于通道的失活态抑制HERG钾电流,但不影响HERG通道蛋白的表达。     

Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain

ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [alpha-(32)P]8-N(3)-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating.     

A model for actin polymerization and the kinetic effects of ATP hydrolysis.

A model for actin polymerization is proposed in which the rate of elongation of actin filaments depends on whether adenosine 5'-triphosphate or adenosine 5'-diphosphate is bound to the two terminal subunits of the filament. This model accounts quantitatively for the experimental data on the kinetics of filament elongation and explains the effect of ATP hydrolysis on actin polymerization.     

R371H和P266T位点突变对ATP敏感性钾通道特性的影响

目的研究R371H和P266T两个位点突变对ATP敏感性钾通道特性的影响，揭示上述位点突变导致心律失常的机制。方法用人胚肾细胞表达Kit6．2野生型、R371H和P266T位点突变后的通道，用膜片钳技术研究突变后胞内pH对ATP敏感性变构调节的变化。结果野生型、P266T、R371H均成功记录到内向整流性K^＋电流。暴露于不同的pH中时，野生型通道的半数电流抑制ATP浓度（IC50）在pH6．8时较pH7．4时显著增大（70VS22μmoL／L，P〈0．05），ATP浓度-电流曲线显著右移；R371H和P266T的IC50在pH6．8与pH7．4时差异无统计学意义。结论胞内pH对ATP敏感性变构调节的丧失，可能是R371H和P266T位点突变时导致心律失常的重要机制。     

Responses of Cremasteric Arterioles of Spontaneously Hypertensive Rats to Changes in Extracellular K+ Concentration

Objective : The goal of this study was to determine the effect of changes in extracellular K⁺ concentration ([K⁺]₀) on active tone in cremasteric arterioles of spontaneously hypertensive rats (SHR) and their normotensive Wistar-Kyoto (WKY) and Wistar controls. Methods : Diameters of third- and fourth-order arterioles were measured in the cremaster muscle of hypertensive and normotensive rats during abrupt changes in superfusate K⁺ concentration from 4.7 mM to 0 mM to 15 mM K⁺. Results : Arterioles constricted in response to superfusion with 0 mM K⁺ and exhibited a large, transient dilation in response to an abrupt change from 0 mM to 15 mM [K⁺]_o. Arteriolar dilation in response to 15 mM K⁺ was significantly larger in 12–15-week-old SHR than in WKY or Wistar controls. Arteriolar responses to 15 mM K⁺ were not significantly different in 4–6-week-old SHR and WKY. Dilator responses to 15 mM K⁺ were generally inhibited by 1 mM ouabain, although ouabain was less effective in inhibiting 15 mM K⁺-induced dilation in arterioles of SHR and WKY than in Wistar rats. Conclusions : Dilation of cremasteric arterioles in response to 15 mM [K⁺]₀, is mediated, at least in part, via stimulation of the electrogenic Na⁺/K⁺ pump, although Na⁺/K⁺-pump-independent components may also contribute to the response. Arterioles of SHR with established hypertension exhibit an altered response to elevated [K⁺]₀ which is not present in SHR in the early stage of hypertension.     

乙醇及其代谢产物对新生大鼠原代培养心房肌细胞乙酰胆碱敏感型钾通道Kir3.1蛋白表达的影响

目的 明确乙醇及其代谢产物乙醛对新生SD大鼠原代心房肌细胞乙酰胆碱敏感型钾通道Kir3.1蛋白表达的影响,并探讨该通道及乙醛在急性乙醇中毒引发心律失常的过程中所发挥的效应.方法 采用胰蛋白酶及Ⅱ型胶原酶提取并培养150只新生SD大鼠原代心房肌细胞,并采用免疫荧光法进行肌钙蛋白Ⅰ鉴定.采用细胞计数套件-8(CCK-8)法分别测定200 ～ 800 mmol/L乙醇及50～ 500 μmol/L乙醛处理24 h后细胞的生存率,以确定导致新生SD大鼠原代心房肌细胞凋亡所需的乙醛及乙醇浓度.建立以最大非凋亡浓度(200 mmol/L)的乙醇处理24h及相应剂量(100 μmol/L)乙醛处理24 h的新生SD大鼠原代心房肌细胞模型.采用Western blot法检测乙醇处理组、乙醛处理组及对照组心房肌细胞Kir3.1蛋白的表达情况,并对结果进行统计学分析.结果 (1)成功培养新生SD大鼠原代心房肌细胞,细胞免疫荧光鉴定所培养细胞为心肌细胞,且90％以上细胞肌钙蛋白Ⅰ染色阳性.(2) CCK-8法测定显示400 mmol/L以上的乙醇处理组的心肌细胞生存率明显低于对照组(P＜0.05),而200 mmol/L及以下的乙醇处理组心肌细胞生存率与对照组相比差异无统计学意义(P ＞0.05);400 μmol/L以上的乙醛处理组的心肌细胞生存率明显低予对照组(P＜0.05),而350 μmol/L及以下的乙醛处理组心肌细胞生存率与对照组相比差异无统计学意义(P＞0.05).(3)Western blot检测显示200 mmol/L乙醇及100 μmol/L的乙醛处理24h的新生SD大鼠原代房心肌细胞Kir3.1蛋白表达分别高于对照组(44.52±23.07)％及(45.04±22.01)％ (P ＜0.01),而乙醇组与乙醛组之间Kir3.1蛋白表达差异无统计学意义(P＞0.05).结论 急性乙醇及乙醛处理均能够明显上调乙酰胆碱敏感型钾通道Kir3.1蛋白的表达.     

Beneficial effects of adenosine triphosphate-sensitive K+ channel opener on liver ischemia/reperfusion injury

AIM: To investigate the effect of diazoxide administration on liver ischemia/reperfusion injury.METHODS: Wistar male rats underwent partial liver ischemia performed by clamping the pedicle from the medium and left anterior lateral segments for 1 h under mechanical ventilation. They were divided into 3 groups: Control Group, rats submitted to liver manipulation, Saline Group, rats received saline, and Diazoxide Group, rats received intravenous injection diazoxide (3.5 mg/kg) 15 min before liver reperfusion. 4 h and 24 h after reperfusion, blood was collected for determination of aspartate transaminase (AST), alanine transaminase (ALT), tumor necrosis factor (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), nitrite/nitrate, creatinine and tumor growth factor-β1 (TGF-β1). Liver tissues were assembled for mitochondrial oxidation and phosphorylation, malondialdehyde (MDA) content, and histologic analysis. Pulmonary vascular permeability and myeloperoxidase (MPO) were also determined.RESULTS: Four hours after reperfusion the diazoxide group presented with significant reduction of AST (2009 ± 257 U/L vs 3523 ± 424 U/L, P = 0.005); ALT (1794 ± 295 U/L vs 3316 ± 413 U/L, P = 0.005); TNF-α (17 ± 9 pg/mL vs 152 ± 43 pg/mL, P = 0.013; IL-6 (62 ± 18 pg/mL vs 281 ± 92 pg/mL); IL-10 (40 ± 9 pg/mL vs 78 ± 10 pg/mL P = 0.03), and nitrite/nitrate (3.8 ± 0.9 μmol/L vs 10.2 ± 2.4 μmol/L, P = 0.025) when compared to the saline group. A significant reduction in liver mitochondrial dysfunction was observed in the diazoxide group compared to the saline group (P < 0.05). No differences in liver MDA content, serum creatinine, pulmonary vascular permeability and MPO activity were observed between groups. Twenty four hours after reperfusion the diazoxide group showed a reduction of AST (495 ± 78 U/L vs 978 ± 192 U/L, P = 0.032); ALT (335 ± 59 U/L vs 742 ± 182 U/L, P = 0.048), and TGF-β1 (11 ± 1 ng/mL vs 17 ± 0.5 ng/mL, P = 0.004) serum levels when compared to the saline group. The control group did not present alterations when compared to the diazoxide and saline groups.CONCLUSION: Diazoxide maintains liver mitochondrial function, increases liver tolerance to ischemia/reperfusion injury, and reduces the systemic inflammatory response. These effects require further evaluation for using in a clinical setting.     

Role of K+ATP Channels in Ischemic Preconditioning and Cardioprotection

Summary. Since the phenomenon of ischemic preconditioning was first described some 15 years ago, interest in strategies aimed at reducing infarct size has increased. During the past 10 years, investigations into the mechanism of ischemic preconditioning have clearly demonstrated the cardioprotective effect of K⁺ _ATP channel opening. Thus, K⁺ _ATP channel activation has been shown to be involved in this protection by a variety of stimuli, including a brief period of complete ischemia (classic ischemic preconditioning) or a partial coronary artery occlusion. In addition, ischemia in remote organs and nonischemic stimuli in the heart such as ventricular pacing, stretch, and heat stress also confer protection via K⁺ _ATP channel activation. Pharmacological agents that open K⁺ _ATP channels reduce infarct size, but K⁺ _ATP channel opening must occur prior to or early during the sustained infarct-producing coronary artery occlusion, while the degree and memory of cardioprotection are less than those produced by classic ischemic preconditioning. Although the exact mechanism by which K⁺ _ATP channel activation protects is still incompletely understood, recent studies indicate a role for the mitochondrial K⁺ _ATP channels. Before K⁺ _ATP channel opening can be employed in patients at increased risk of developing myocardial infarction (e.g., unstable angina), it is mandatory to determine whether tolerance (tachyphylaxia) occurs with repeated administration of K⁺ _ATP channel openers in a fashion similar to what occurs with ischemic preconditioning.     

ATP-sensitive K+ channels of vascular smooth muscle cells

ATP-sensitive potassium channels (K(ATP)) of vascular smooth muscle cells represent potential therapeutic targets for control of abnormal vascular contractility. The biophysical properties, regulation and pharmacology of these channels have received intense scrutiny during the past twenty years, however, the molecular basis of vascular K(ATP) channels remains ill-defined. This review summarizes the recent advancements made in our understanding of the molecular composition of vascular K(ATP) channels with a focus on the evidence that hetero-octameric complexes of Kir6.1 and SUR2B subunits constitute the vascular K(ATP) subtype responsible for control of arterial diameter by vasoactive agonists.